Proteases from gram positive organisms

ABSTRACT

The present invention relates to the identification of a novel metalloprotease in gram positive microorganisms. The present invention provides the nucleic acid and amino acid sequences for the metalloprotease. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding the metalloprotease. The present invention provides host cells which further comprises a nucleic acid encoding desired heterologous proteins such as enzymes. The present invention also provides cleaning compositions, animal feeds and compositions used to treat a textile that include the metalloprotease of the present invention.

FIELD OF THE INVENTION

The present invention relates to metalloproteases derived from grampositive microorganisms. The present invention provides nucleic acid andamino acid sequences of a metalloproteases identified in Bacillus. Thepresent invention also provides methods for the production of themetalloprotease in host cells as well as the production of heterologousproteins in a host cell having a mutation or deletion of part or all ofthe metalloprotease of the present invention.

BACKGROUND OF THE INVENTION

Gram positive microorganisms, such as members of the group Bacillus,have been used for large-scale industrial fermentation due, in part, totheir ability to secrete their fermentation products into the culturemedia. In gram positive bacteria, secreted proteins are exported acrossa cell membrane and a cell wall, and then are subsequently released intothe external media usually maintaining their native conformation.

Various gram positive microorganisms are known to secrete extracellularand/or intracellular proteases at some stage in their life cycles. Someof these proteases are produced in large quantities for industrialpurposes. However, a negative aspect of the presence of proteases ingram positive organisms is their contribution to the overall degradationof secreted heterologous or foreign proteins.

The classification of proteases founding microorganisms is based ontheir catalytic mechanism which results in four groups: serineproteases, metalloproteases, cysteine proteases, and aspartic proteases.These categories can be distinguished by their sensitivity to variousinhibitors. For example, serine proteases are inhibited byphenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate(DIFP); metalloproteases by chelating agents; cysteine proteases byiodoacetamide and heavy metals and aspartic proteases by pepstatin.Further, in general, serine proteases have alkaline pH optima,metalloproteases are optimally active around neutrality, and cysteineand aspartic proteases have acidic pH optima (Biotechnology Handbooks,Bacillus. Vol. 2, edited by Harwood, 1989, Plenum Press, New York).

Metalloproteases are the most diverse of the catalytic types ofproteases. About half of the families comprise enzymes containing theHis-Glu-Xaa-Xaa-His (or HEXXH) motif which has been shown by X-raycrystallography to form part of the site for binding of the metal atom,commonly zinc. In at least one family of metalloproteases, a glutamicacid residue completes the metal-binding site, HEXXH +E. For example,the most well characterized of the metalloproteases, thermolysin,contains this motif. The three dimensional structure of thermolysinshows that, in the HEXXH motif, the His residues are zinc ligands andthe Glu residue has a catalytic function. (Methods in Enzymology, Vol.248, Academic Press, Inc., 1994).

An interesting variation of the HEXXH +E motif can be found in themetalloprotease family, m16, in which this motif is inverted and seen asHXXEH +E. Members of this family include pitrilysin (Methods inEnzymology, Vol. 248, Academic Press, Inc., 1994, pp. 684-692) andinsulinase or insulypin (Methods in Enzymology, Vol. 248, AcademicPress, Inc., 1994, pp. 211-215).

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a heretofore unknownmetalloprotease (MP) found in gram positive microorganisms, uses of theMP in industrial applications, and advantageous strain improvementsbased on genetically engineering such microorganisms to delete,underexpress or overexpress that MP. The present invention is based uponthe discovery that MP has overall amino acid relatedness to Escherichiacoli pitrilysin.

The present invention is based upon Applicant's discovery that theinverted version of the characteristic metalloprotease amino acid motifHXXEH +E and putative transmembrane domains exist in Bacillus subtilisMP. Applicant's discovery, in addition to providing a new and usefulprotease and methods of detecting DNA encoding other such proteases in agram positive microorganism, provides several advantages which mayfacilitate optimization and/or modification of strains of gram positivemicroorganisms, such as Bacillus, for expression of desired, e.g.heterologous, proteins. Such optimizations, as described below indetail, allow the construction of strains having decreased proteolyticdegradation of desired expression products.

Due to the relatedness of MP to pitrilysin and insulysin, zincmetalloendopeptidases which have been shown to degrade small peptides ofless than 7 kd such as glucagon and insulin, it can be concluded that MPis also an endopeptidase and would be expected to behave similarly topitrilysin and insulysin.

The present invention encompasses the naturally occurring MP encoded bynucleic acid found in a Bacillus species as well as the nucleic acid andamino acid molecules having the sequences disclosed in SEQ ID NOS: 1 end2. In one embodiment, the gram positive microorganism is a Bacillus. Ina further embodiment, the Bacillus is preferably selected from the groupconsisting of Bacillus subtilis, Bacillus stearothermophilus, Bacilluslicheniformis and Bacillus amyloliquefaciens. The invention furtherprovides for a metalloprotease that has at least 80%, preferably atleast 90%, most preferably 95% homology with the amino acid sequence ofSEQ ID NO: 2. The invention also provides for a nucleic acid whichencodes a metalloprotease that has at least 80%, preferably at least90%, most preferably 95% homology with the nucleotide sequence shown inSEQ ID NO:1.

In a preferred embodiment, the present invention encompasses thenaturally occurring MP nucieic acid molecule having the sequence foundin Bacillus subtilis I-168 strain (Bacillus Genetic Stock Center,accession number 1A1, Columbus, Ohio) in the region of about 1757 kbfrom the point of origin. In another preferred embodiment, the Bacillussubtilis MP nucleic acid and amino acid molecules have the sequences asshown in FIGS. 1A-1F (SEQ ID NOS:1 and 2).

The present invention provides isolated polynucleotide and amino acidsequences for Bacillus subtilis MP in FIGS. 1A-1F (SEQ ID NOS:1 and 2).Due to the degeneracy of the genetic code, the present inventionencompasses any nucleic acid sequence that encodes the Bacillus subtilisMP amino acid sequence. The present invention provides expressionvectors and host cells comprising a nucleic acid encoding a grampositive MP. The present invention also provides methods of making thegram positive MP.

The present invention encompasses novel amino acid variations of grampositive MP amino acid sequences disclosed herein that have proteolyticactivity. Naturally occurring gram positive MP as well asproteolytically active amino acid variations or derivatives thereof,have application in the textile industry, in cleaning compositions andin animal feed.

The present invention also encompasses amino acid variations orderivatives of gram positive MP that do not have the characteristicproteolytic activity as long as the nucleic acid sequences encoding suchvariations or derivatives would have sufficient 5′ and 3′ coding regionsto be capable of being integrated into a gram positive organism genome.Such variants would have applications in gram positive expressionsystems where it is desirable to delete, mutate, alter or otherwiseincapacitate the naturally occurring metalloprotease in order todiminish or delete its proteolytic activity. Such an expression systemwould have the advantage of allowing for greater yields of recombinantheterologous proteins or polypeptides.

The present invention provides methods for detecting gram positivemicroorganism homologues of B. subtilis MP that comprises hybridizingpart or all of the nucleic acid encoding B. subtilis MP with nucleicacid derived from gram positive organisms, either of genomic or cDNAorigin. Accordingly, the present invention provides a method fordetecting a gram positive microorganism MP, comprising the steps ofhybridizing gram positive microorganism nucleic acid under lowstringency conditions to a probe, wherein the probe comprises part orall of the nucleic acid sequence shown in FIGS. 1A-1F (SEQ ID NO:1); andisolating the gram positive nucleic acid which hybridizes to said probe.

The production of desired heterologous proteins or polypeptides in grampositive microorganisms may be hindered by the presence of one or moreproteases which degrade the produced heterologous protein orpolypeptide. One advantage of the present invention is that it providesmethods and expression systems which can be used to prevent thatdegradation, thereby enhancing yields of the desired heterologousprotein or polypeptide.

Accordingly, the present invention provides a gram positivemicroorganism that can be used as a host cell having a mutation ordeletion of part or all of the gene encoding MP, which results in theinactivation of the MP proteolytic activity, either alone or incombination with mutations in other proteases, such as apr, npr, epr,mpr, bpf or isp, or other proteases known to those of skill in the art.In one embodiment of the present invention, the gram positivemicroorganism is a member of the genus Bacillus. In a preferredembodiment, the Bacillus is selected from the group consisting of B.subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus,B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and B. thuringiensis. In a further preferred embodiment, theBacillus is Bacillus subtilis.

In another aspect, the gram positive host cell having one or moremetalloprotease deletions or mutations is further genetically engineeredto produce a desired protein. In one embodiment of the presentinvention, the desired protein is heterologous to the gram positive hostcell. In another embodiment, the desired protein is homologous to thehost cell.

In another embodiment, a host cell is engineered to produce MP. The grampositive microorganism may be normally sporulating or non-sporulating.In a preferred embodiment, the gram positive host cell is a Bacillus. Inanother embodiment, the Bacillus is selected from the group consistingof B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans,B. circulans, B. lautus and b. thuringiensis. In a further preferredembodiment, the Bacillus host cell is Bacillus subtilis.

In a further aspect of the present invention, gram positive MP isproduced on an industrial fermentation scale in a microbial hostexpression system. In another aspect, isolated and purified recombinantMP is used in compositions intended for use in the textile industry, incleaning compositions, such as detergents, and in animal feeds.Accordingly, the present invention provides a cleaning composition,animal feed and a composition for the treatment of a textile comprisingMP. The metalloprotease, MP, may be used alone or in combination withother enzymes and/or mediators or enhancers.

As noted, the present invention provides a cleaning compositioncomprising a metalloprotease, MP, comprising the amino acid sequenceshown in SEQ ID NO:2. Also provided are cleaning compositions comprisinga metallaprotease having at least 80%; preferably 90%, more preferably95% homology with the amino acid sequence shown in SEQ ID NO:2 oroomprising a metalloprotease encoded by a gene that hybridizes with thenucleic acid shown in SEQ ID NO:1.

Further there is provided an animal feed comprising a metalloprotease,MP, comprising the amino acid sequence shown in SEQ ID NO:2. Alsoprovided are animal feeds comprising a metalloprotease having at least80%, preferably 90%, more preferably 95% homology with the amino acidsequence shown in SEQ ID NO:2 or comprising a metalloprotease encoded bya gene that hybridizes with the nucleic acid shown in SEQ ID NO:1.

Also provided is a composition for the treatment of a textile comprisinga metalloprotease, MP, comprising the amino acid sequence shown in SEQID NO:2. Also provided are compositions for the treatment of a textilecomprising a metalloprotease having at least 80%, preferably 90%, morepreferably 95% homology with the amino acid sequence shown in SEQ IDNO:2 or comprising a metalloproteaseencoded by a gene that hybridizeswith the nucleic acid shown in SEQ ID NO:1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the DNA and amino acid sequence for Bacillus subtilisMP (YmfH) (SEQ ID NO:1).

FIGS. 2A-2B show an amino acid alignment of Escherichia coli pitrilysinand Bacillus subtilis MP (YmfH). The amino acid motif HXXEH +E is notedin FIGS. 2A-2B.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

As used herein, the genus Bacillus includes all members known to thoseof skill in the art, including but not limited to B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and B. thuringiensis.

The present invention relates to a newly characterized metalloprotease(MP) from gram positive organisms. In a preferred embodiment, the grampositive organisms is a Bacillus. In another preferred embodiment, theBacillus is selected from the group consisting of B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and B. thuringiensis.

In another preferred embodiment, the gram positive organism is Bacillussubtilis and MP has the amino acid sequence encoded by the nucleic acidmolecule having the sequence that occurs around 1757 kilobases from thepoint of origin of Bacillus subtilis I-168.

In another preferred embodiment, MP has the nucleic acid and amino acidsequence as shown in FIGS. 1A-1F (SEQ ID NOS: 1 and 2). The presentinvention encompasses the use of amino acid variations of the amino addsequences disclosed in FIGS. 1A-1D (SEQ ID NO: 2) that have proteolyticactivity. Such proteolytic amino acid variants can be used in thetextile industry, animal feed and cleaning compositions. The presentinvention also encompasses the use of B. subtilis amino acid variationsor derivatives that are not proteolytically active. DNA encoding suchvariants can be used in methods designed to delete or mutate thenaturally occurring host cell MP.

As used herein, “nucleic acid” refers to a nucleotide or polynucleotidesequence, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Asused herein “amino acid” refers to peptide or protein sequences orportions thereof. A “polynucleotide homologue” as used herein refers toa gram positive microorganism polynucleotide that has at least 80%,preferably at least 90% and more preferably at least 95% identity toB.subtilis MP, or which is capable of hybridizing to B. subtilis MPunder conditions of high stringency and which encodes an amino acidsequence having metalloprotease activity.

The terms “isolated” or “purified” as used herein refer to a nucleicacid or amino acid that is removed from at least one component withwhich it is naturally associated.

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in the chosen gram positivehost cell. Examples of heterologous proteins include enzymes such ashydrolases including proteases, cellulases, carbohydrases such asamylases, and lipases; isomerases such as racemases, epimerases,tautomerases, or mutases; oxidases, reductases, transferases, kinasesand phophatases. The heterologous gene may encode therapeuticallysignificant proteins or peptides, such as growth factors, cytokines,ligands, receptors and inhibitors, as well as vaccines and antibodies.The gene may encode commercially important industrial proteins orpeptides, such as proteases, carbohydrases such as amylases andglucoamylases, cellulases, oxidases and lipases. The gene of interestmay be a naturally occurring gene, a mutated gene or a synthetic gene.

The term “homologous protein” refers to a protein or polypeptide nativeor naturally occurring in the chosengram positive host cell. Theinvention includes host cells producing the homologous protein viarecombinant DNA technology. The present invention encompasses a grampositive host cell having a deletion or interruption of the nucleic acidencoding the naturally occurring homologous protein, such as a protease,and having nucleic acid encoding the homologous protein re-introduced ina recombinant form. In another embodiment, the host cell produces thehomologous protein.

As used herein, the term “overexpressing” when referring to theproduction of a protein in a host cell means that the protein isproduced in greater amounts than in its naturally occurring environment.

As used herein, the phrase “proteolytic activity” refers to a proteinthat is able to hydrolyze a peptide bond. Enzymes having proteolyticactivity are described in Enzyme Nomenclature, 1992, edited WebbAcademic Press, Inc.

The unexpected discovery of the metalloprotease MP found in translateduncharacterised B.subtilis genomic sequences provides a basis forproducing host cells, expression methods and systems which can be usedto prevent the degradation of recombinantly produced heterologousproteins.

Accordingly, in a preferred embodiment, the host cell is a gram positivehost cell that has a deletion or mutation in the naturally occurringnucleic acid encoding MP said mutation resulting in deletion orinactivation of the production by the host cell of the MP proteolyticgene product. The host cell may additionally be genetically engineeredto produced a desired protein or polypeptide.

It may also be desired to genetically engineer host cells of any type toproduce a gram positive metalloprotease. Such host cells are used inlarge scale fermentation to produce large quantities of themetalloprotease which may be isolated or purified and used in cleaningproducts, such as detergents.

I. Metalloprotease Sequences

The present invention encompasses the use of MP polynucleotidehomologues encoding gram positive microorganism metalloproteases MPwhich have at least 80%, preferably at least 90%, more preferably atleast 95% identity to B. subtilis MP as long as the homologue encodes aprotein that has proteolytic activity. A preferred MP polynucleotidehomologue has 96% homology to B. subtilis MP.

Gram positive polynucleotide homologues of B. subtilis MP may beobtained by standard procedures known in the art from, for example,cloned DNA (e.g., a DNA “library”), genomic DNA libraries, by chemicalsynthesis once identified, by cDNA cloning, or by the cloning of genomicDNA, or fragments thereof, purified from a desired cell. (See, forexample, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press,Ltd., Oxford, U.K. Vol. I, II.) A preferred source is from genomic DNA.

As will be understood by those of skill in the art, the polynucleotidesequence and amino acid sequence disclosed in FIGS. 1A-1F may reflectinadvertent errors inherent to nucleic acid sequencing technology. Thepresent invention encompasses the naturally occurring nucleic acidmolecule having the nucleic acid sequence obtained from the genomicsequence of Bacillus species.

Nucleic acid encoding Bacillus subtilis MP starts around 1757 kilobasescounting from the point of origin in the Bacillus subtilis strain I-168(Anagnostopala, 1961, J. Bacteriol., 81:741-746 or Bacillus GenomicStock Center, accession 1A1, Columbus, Ohio). The Bacillus subtilispoint of origin has been described in Ogasawara, N. (1995, Microbiology141:Pt.2 257-59). Bacillus subtilis MP has a length of 415 amino acids.Based upon the location of the DNA encoding Bacillus subtilis MP,naturally occurring B. subtilis MP can be obtained by methods known tothose of skill in the art including PCR technology.

Oligonucleotide sequences or primers of about 10-30 nucleotides inlength can be designed from the polynucleotide sequence disclosed inFIGS. 1A-1F and used in PCR technology to isolate the naturallyoccurring sequence from B. subtilis genomic sequences.

Another general strategy for the “cloning” of B. subtilis genomic DNApieces for sequencing uses inverse PCR. A known region is scanned for aset of appropriate restriction enzyme cleavage sites and inverse PCR isperformed with a set of DNA primers determined from the outermost DNAsequence. The DNA fragments from the inverse PCR are directly used astemplate in the sequencing reaction. The newly derived sequences can beused to design new oligonucleotides. These new oligonucleotides are usedto amplify DNA fragments with genomic DNA as template. The sequencedetermination on both strands of a DNA region is finished by applying aprimer walking strategy on the genomic PCR fragments. The benefit ofmultiple starting points in the primer walking results from the seriesof inverse PCR fragments with different sizes of new “cloned” DNApieces. From the most external DNA sequence, a new round of inverse PCRis started. The whole inverse PCR strategy is based on the sequentialuse of conventional taq polymerase and the use of long range inverse PCRin those cases in which the taq polymerase failed to amplify DNAfragments. Nucleic acid sequencing is performed using standardtechnology. One method for nucleic acid sequencing involves the use of aPerkin-Elmer Applied Biosystems 373 DNA sequencer (Perkin-Elmer, FosterCity, Calif.) according to manufacturers instructions.

Nucleic acid sequences derived from genomic DNA may contain regulatoryregions in addition to coding regions. Whatever the source, the isolatedMP gene should be molecularly cloned into a suitable vector forpropagation of the gene.

In molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the MP may be accomplished in a number of ways. Forexample, a B. subtilis MP gene of the present invention or its specificRNA, or a fragment thereof, such as a probe or primer, may be isolatedand labeled and then used in hybridization assays to detect a grampositive MP gene. (Benton, W. and Davis, R., 1977, Science 196:180;Grunstein, M. and Hogness, D., 1975, Proc. Natl. Aced. Sci. USA72:3961). Those DNA fragments sharing substantial sequence similarity tothe probe will hybridize under stringent conditions.

Accordingly, the present invention provides a method for the detectionof gram positive MP polynucleotide homologues which compriseshybridizing part or all of a nucleic acid sequence of B. subtilis MPwith gram positive microorganism nucleic acid of either genomic or cDNAorigin.

Also included within the scope of the present invention is the use ofgram positive microorganism polynucleotide sequences that are capable ofhybridizing to the nucleotide sequence of B. subtilis MP underconditions of intermediate to maximal stringency. Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex, as taught in Berger and Kimmel (1987, Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol. 152, AcademicPress, San Diego, Calif.) incorporated herein by reference, and confer adefined “stringency” as explained below.

“Maximum stringency” typically occurs at about Tm-5° C. (5° C. below theTm of the probe); “high stringency” at about 5° C. to 10° C. below Tm;“intermediate stringency” at about 10° C. to 20° C. below Tm; and “lowstringency” at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate or low stringency hybridization can be used to identify ordetect polynucleotide sequence homologues.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs, J., (1994), Dictionary of Biotechnology, StocktonPress, New York, N.Y.).

The process of amplification as carried out in polymerase chain reaction(PCR) technologies is described in Dieffenbach, C W and G S Dveksler,(PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview,N.Y., 1995). A nucleic acid sequence of at least about 10 nucleotidesand as many as about 60 nucleotides from B. subtilis MP, preferablyabout 12 to 30 nucleotides, and more preferably about 20-25 nucleotidescan be used as a probe or PCR primer.

The B. subtilis MP amino acid sequences (shown in FIGS. 1A-1F) wereidentified via a BLAST search (Atschul, Stephen, Basic local alignmentsearch tool, J. Mol. Biol., 215:403-410) of Bacillus subtilis genomicnucleic acid sequences. B. subtilis MP (YmfH) was identified by itsoverall nucleic acid identity to the metalloprotease, pitrilysin fromEscherichia coli, including the presence of the catalytic domain HXXEH+E as shown in FIGS. 2A-2B.

II. Expression Systems

The present invention provides host cells, expression methods andsystems for the enhanced production and secretion of desiredheterologous or homologous proteins in gram positive microorganisms. Inone embodiment, a host cell is genetically engineered to have a deletionor mutation in the gene encoding a gram positive MP such that therespective activity is deleted. In another embodiment of the presentinvention, a gram positive microorganism is genetically engineered toproduce and/or overproduce a metalloprotease of the present invention.

Inactivation of a Gram Positive Metalloprotease in a Host Cell

Producing an expression host cell incapable of producing the naturallyoccurring metalloprotease necessitates the replacement and/orinactivation of the naturally occurring gene in the genome of the hostcell. In a preferred embodiment, the mutation is a non-+revertingmutation.

One method for mutating a nucleic acid encoding a gram positivemetalloprotease is to clone the nucleic acid or part thereof, modify thenucleic acid by site directed mutagenesis and reintroduce the mutatednucleic acid into the cell on a plasmid. By homologous recombination,the mutated gene can be introduced into the chromosome. In the parenthost cell, the result is that the naturally occurring nucleic acid andthe mutated nucleic acid are located in tandem on the chromosome. Aftera second recombination, the modified sequence is left in the chromosomehaving thereby effectively introduced the mutation into the chromosomalgene for progeny of the parent host cell.

Another method for inactivating the metalloprotease proteolytic activityis through deleting the chromosomal gene copy. In a preferredembodiment, the entire gene is deleted, the deletion occurring in suchas way as to make reversion impossible. In another preferred embodiment,a partial deletion is produced, provided that the nucleic acid sequenceleft in the chromosome is too short for homologous recombination with aplasmid encoding the metalloprotease gene. In another preferredembodiment, nucleic acid encoding the catalytic amino acid residues aredeleted.

Deletion of the naturally occurring gram positive microorganismmetalloprotease can be carried out as follows. A metalloprotease geneincluding its 5′ and 3′ regions is isolated and inserted into a cloningvector. The coding region of the metalloprotease gene is deleted fromthe vector in vitro, leaving behind a sufficient amount of the 5′ and 3′flanking sequences to provide for homologous recombination with thenaturally occurring gene in the parent host cell. The vector is thentransformed into the gram positive host cell. The vector integrates intothe chromosome via homologous recombination in the flanking regions.This method leads to a gram positive strain in which the protease genehas been deleted.

The vector used in an integration method is preferably a plasmid. Aselectable marker may be included to allow for ease of identification ofdesired recombinant microorganisms. Additionally, as will be appreciatedby one of skill in the art, the vector is preferably one which can beselectively integrated into the chromosome. This can be achieved byintroducing an inducible origin of replication, for example, atemperature sensitive origin into the plasmid. By growing thetransformants at a temperature to which the origin of replication issensitive, the replication function of the plasmid is inactivated,thereby providing a means for selection of chromosomal integrants.Integrants may be selected for growth at high temperatures in thepresence of the selectable marker, such as an antibiotic. Integrationmechanisms are described in WO 88/06623.

Integration by the Campbell-type mechanism can take place in the 5′flanking region of the protease gene, resulting in a protease positivestrain carrying the entire plasmid vector in the chromosome in themetalloprotease locus. Since illegitimate recombination will givedifferent results, it will be necessary to determine whether thecomplete gene has been deleted, such as through nucleic acid sequencingor restriction maps.

Another method of inactivating the naturally occurring metalloproteasegene is to mutagenize the chromosomal gene copy by transforming a grampositive microorganism with oligonucleotides which are mutagenic.Alternatively, the chromosomal metalloprotease gene can be replaced witha mutant gene by homologous recombination.

The present invention encompasses host cells having additional proteasedeletions or mutations, such as deletion of or mutation(s) in the genesencoding apr, npr, epr, mpr and others known to those of skill in theart.

One assay for the detection of mutants involves growing the Bacillushost cell on medium containing a protease substrate and measuring theappearance or lack thereof, of a zone of clearing or halo around thecolonies. Host cells which have an inactive protease will exhibit littleor no halo around the colonies.

III. Production of Metalloprotease

For production of metalloprotease in a host cell, an expression vectorcomprising at least one copy of nucleic acid encoding a gram positivemicroorganism MP, and preferably comprising multiple copies, istransformed into the host cell under conditions suitable for expressionof the metalloprotease. In accordance with the present invention,polynucleotides which encode a gram positive microorganism MP, orfragments thereof, or fusion proteins or polynucleotide homologuesequences that encode amino acid variants of B. subtilis MP, may be usedto generate recombinant DNA molecules that direct their expression inhost cells. In a preferred embodiment, the gram positive host cellbelongs to the genus Bacillus. In a further preferred embodiment, thegram positive host cell is B. subtilis.

As will be understood by those of skill in the art, it may beadvantageous to produce polynucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particular grampositive host cell (Murray, E. et al., (1989), Nuc. Acids Res.,17:477-508) can be selected, for example, to increase the rate ofexpression or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, than transcripts produced from anaturally occurring sequence.

Altered MP polynucleotide sequences which may be used in accordance withthe invention include deletions, insertions or substitutions ofdifferent nucleotide residues resulting in a polynucleotide that encodesthe same or a functionally equivalent MP homologue, respectively. Asused herein a “deletion” is defined as a change in the nucleotidesequence of the MP resulting in the absence of one or more amino acidresidues.

As used herein, an “insertion” or “addition” is that change in thenucleotide sequence which results in the addition of one or more aminoacid residues as compared to the naturally occurring MP.

As used herein, “substitution” results from the replacement of one ormore nucleotides or amino acids by different nucleotides or amino acids,respectively. The change(s) in the nucleotides(s) can either result in achange in the amino acid sequence or not.

The encoded protein may also show deletions, insertions or substitutionsof amino acid residues which produce a silent change and result in afunctionally,equivalent MP variant. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the variant retains its proteolytic ability. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine;glycine, alanine; asparagine, glutamine; serine, threonine,phenylalanine, and tyrosine.

The MP polynucleotides of the present invention may be engineered inorder to modify the cloning, processing and/or expression of the geneproduct. For example, mutations may be introduced using techniques whichare well known in the art, i.e., site-directed mutagenesis to insert newrestriction sites, to alter glycosylation patterns or to change codonpreference, for example.

In one embodiment of the present invention, a gram positivemicroorganism MP polynucleotide may be ligated to a heterologoussequence to encode a fusion protein. A fusion protein may also beengineered to contain a cleavage site located between themetalloprotease nucleotide sequence and the heterologous proteinsequence, so that the metalloprotease may be cleaved and purified awayfrom the heterologous moiety.

IV. Vector Sequences

Expression vectors used in expressing the metalloproteases of thepresent invention in gram positive microorganisms comprise at least onepromoter associated with MP, which promoter is functional in the hostcell. In one embodiment of the present invention, the promoter is thewild-type promoter for the selected metalloprotease and in anotherembodiment of the present invention, the promoter is heterologous to themetalloprotease, but still functional in the host cell. In one preferredembodiment of the present invention, nucleic acid encoding themetalloprotease is stably integrated into the microorganism genome.

In a preferred embodiment, the expression vector contains a multiplecloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the term“selectable marker” refers to a gene capable of expression in the grampositive host which allows for ease of selection of those hostscontaining the vector. Examples of such selectable markers include butare not limited to antibiotics, such as, erythromycin, actinomycin,chloramphenicol and tetracycline.

V. Transformation

A variety of host cells can be used for the production Bacillus subtilisMP or MP homologues including bacterial, fungal, mammalian and insectscells. General transformation procedures are taught in Current ProtocolsIn Molecular Biology, (Vol. 1, edited by Ausubel et al., John Wiley &Sons, Inc., 1987, Chapter 9) and include calcium phosphate methods,transformation using DEAE-Dextran and electroporation. Planttransformation methods are taught in Rodriquez (WO 95/14099, published26 May 1995).

In a preferred embodiment, the host cell is a gram positivemicroorganism and in another preferred embodiment, the host cell isBacillus. In one embodiment of the present invention, nucleic acidencoding one or more metalloprotease(s) of the present invention isintroduced into a host cell via an expression vector capable ofreplicating within the Bacillus host cell. Suitable replicating plasmidsfor Bacillus are described in Molecular Biological Methods for Bacillus,Ed. Harwood and Cutting, John Wiley & Sons, 1990, hereby expresslyincorporated by reference; see chapter 3 on plasmids. Suitablereplicating plasmids for B. subtilis are listed on page 92.

In another embodiment, nucleic acid encoding a metalloprotease(s) of thepresent invention is stably integrated into the microorganism genome.Preferred host cells are gram positive host cells. Another preferredhost is Bacillus. Another preferred host is Bacillus subtilis. Severalstrategies have been described in the literature for the direct cloningof DNA in Bacillus. Plasmid marker rescue transformation involves theuptake of a donor plasmid by competent cells carrying a partiallyhomologous resident plasmid (Contente et al., Plasmid, 2:555-571 (1979);Haima et al., Mol. Gen. Genet., 223:185-191 (1990); Weinrauch et al., J.Bacteriol., 154(3):1077-1087 (1983); and Weinrauch et al., J.Bacteriol., 169(3):1205-1211 (1987)). The incoming donor plasmidrecombines with the homologous region of the resident “helper” plasmidin a process that mimics chromosomal transformation.

Transformation by protoplast transformation is described for B. subtilisin Chang and Cohen, (1979), Mol. Gen. Genet., 168:111-115; for B.megatenum in Vorobjeva et al., (1980), FEMS Microbiol. Letters,7:261-263; for B. amyloliquefaciens in Smith et al., (1986), Appl. andEnv. Microbiol., 51:634; for B. thulingiensis in Fisher et al., (1981),Arch. Microbiol., 139:213-217; for B. sphaericus in McDonald, (1984), J.Gen. Microbiol., 130:203; and B. larvae in Bakhiet et al., (1985, Appl.Environ. Microbiol. 49:577). Mann et al., (1986, Current Microbiol.,13:131-135) report on transformation of Bacillus protoplasts andHolubova, (1985), Folia Microbiol., 30:97) disclose methods forintroducing DNA into protoplasts using DNA containing liposomes.

VI. Identification of Transformants

Whether a host cell has been transformed with a mutated or a naturallyoccurring gene encoding a gram positive MP, detection of thepresence/absence of marker gene expression can suggest whether the geneof interest is present. However, its expression should be confirmed. Forexample, if the nucleic acid encoding a metalloprotease is insertedwithin a marker gene sequence, recombinant cells containing the insertcan be identified by the absence of marker gene function. Alternatively,a marker gene can be placed in tandem with nucleic acid encoding themetalloprotease under the control of a single promoter. Expression ofthe marker gene in response to induction or selection usually indicatesexpression of the metalloprotease as well.

Alternatively, host cells which contain the coding sequence for ametalloprotease and express the protein may be identified by a varietyof procedures known to those of skill in the art. These proceduresinclude, but are not limited to, DNA-DNA or DNA-RNA hybridization andprotein bioassay or immunoassay techniques which include membrane-based,solution based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

The presence of the metalloprotease polynucleotide sequence can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of B. subtilis MP.

VlI. Assay of Protease Activity

There are various assays known to those of skill in the art fordetecting and measuring protease activity. There are assays based uponthe release of acid-soluble peptides from casein or hemoglobin measuredas absorbance at 280 nm or colorimetrically using the Folin method(Bergmeyer, et al., 1984, Methods of Enzymatic Analysis, Vol. 5,Peptidases, Proteinases and their Inhibitors, Verlag Chemie, Weinheim).Other assays involve the solubilization of chromogenic substrates (Ward,1983, Proteinases, in Microbial Enzymes and Biotechnology, (W. M.Fogarty, ed.), Applied Science, London, pp. 251-317).

VIII. Secretion of Recombinant Proteins

Means for determining the levels of secretion of a heterologous orhomologous protein in a gram positive host cell and detecting secretedproteins include using either polyclonal or monoclonal antibodiesspecific for the protein. Examples include enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA) and fluorescent activated cellsorting (FACS). These and other assays are described, among otherplaces, in Hampton, R. et al., (1990, Serological Methods, a LaboratoryManual, APS Press, St. Paul Minn.) and Maddox, D E et al., (1983, J.Exp. Med., 158:1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic and amino acidassays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway N.J.),Promega (Madison Wis.), and US Biochemical Corp. (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles and the like. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also,recombinant immunoglobulins may be produced as shown in U.S. Pat. No.4,816,567 and incorporated herein by reference.

IX. Purification of Proteins

Gram positive host cells transformed with polynucleotide sequencesencoding heterologous or homologous protein may be cultured underconditions suitable for the expression and recovery of the encodedprotein from cell culture. The protein produced by a recombinant grampositive host cell comprising a mutation or deletion of themetalloprotease activity will be secreted into the culture media. Otherrecombinant constructions may join the heterologous or homologouspolynucleotide sequences to a nucleotide sequence encoding a polypeptidedomain which will facilitate purification of soluble proteins (Kroll, DJ. et al., (1993), DNA Cell Biol., 12:441-53).

Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath, J., (1992), Protein Expr.Purif. 3:263-281), protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp, Seattle Wash.).The inclusion of a cleavable linker sequence such as Factor XA orenterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and the heterologous protein can be used to facilitatepurification.

X. Uses of The Present Invention

MP and Genetically Engineered Host Cells

The present invention provides genetically engineered host cellscomprising mutations, preferably non-revertable mutations, or deletionsin the naturally occurring gene encoding MP such that the proteolyticactivity is diminished or deleted altogether. The host cell may containadditional protease deletions, such as deletions of the mature subtilisnprotease and/or mature neutral protease disclosed in U.S. Pat. No.5,264,366.

In a preferred embodiment, the host cell is further geneticallyengineered to produce a desired protein or polypeptide. In a preferredembodiment, the host cell is a Bacillus. In a further preferredembodiment, the host cell is a Bacillus subtilis.

In an alternative embodiment, a host cell is genetically engineered toproduce a gram positive MP. In a preferred embodiment, the host cell isgrown under large scale fermentation conditions. In another preferredembodiment, the MP is isolated and/or purified and used in the textileindustry, the feed industry and in cleaning compositions such asdetergents.

As noted, MP can be useful in formulating various cleaning compositions.A number of known compounds are suitable surfactants useful incompositions comprising the MP of the invention. These include nonionic,anionic, cationic, anionic or zwitterionic detergents, as disclosed inU.S. Pat. No. 4,404,128 and U.S. Pat. No. 4,261,868. A suitabledetergent formulation is that described in Example 7 of U.S. Pat. No.5,204,015. The art is familiar with the different formulations which canbe used as cleaning compositions. In addition, MP can be used, forexample, in bar or liquid soap applications, dishcare formulations,contact lens cleaning solutions or products, peptide hydrolysis, wastetreatment, textile applications, as fusion-cleavage enzymes in proteinproduction, etc. MP may comprise enhanced performance in a detergentcomposition (as compared to another detergent protease). As used herein,enhanced performance in a detergent is defined as increasing cleaning ofcertain enzyme sensitive stains such as grass or blood, as determined byusual evaluation after a standard wash cycle.

MP can be formulated into known powdered and liquid detergents having pHbetween 6.5 and 12.0 at levels of about 0.01 to about 5% (preferably0.1% to 0.5%) by weight. These detergent cleaning compositions can alsoinclude other enzymes such as known proteases, amylases, cellulases,lipases or endoglycosidases, as well as builders and stabilizers.

The addition of MP to conventional cleaning compositions does not createany special use limitation. In other words, any temperature and pHsuitable for the detergent is also suitable for the present compositionsas long as the pH is within the above range, and the temperature isbelow the described MP's denaturing temperature. In addition, MP can beused in a cleaning composition without detergents, again either alone orin combination with builders and stabilizers.

Proteases can be included in animal feed such as part of animal feedadditives as described in, for example, U.S. Pat. No. 5,612,055; U.S.Pat. No. 5,314,692; and U.S. Pat. No. 5,147,642.

One aspect of the invention is a composition for the treatment of atextile that includes MP. The composition can be used to treat forexample silk or wool as described in publications such as RD 216,034; EP134,267; U.S. Pat. No. 4,533,359; and EP 344,259.

MP Polynucleotides

A B. subtilis MP polynucleotide, or any part thereof, provides the basisfor detecting the presence of gram positive microorganism MPpolynucleotide homologues through hybridization techniques and PCRtechnology.

Accordingly, one aspect of the present invention is to provide fornucleic acid hybridization and PCR probes which can be used to detectpolynucleotide sequences, including genomic and cDNA sequences, encodinggram positive MP or portions thereof.

The manner and method of carrying out the present invention may be morefully understood by those of skill in the art by reference to thefollowing examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto

EXAMPLE I

Preparation of a Genomic library

The following example illustrates the preparation of a Bacillus genomiclibrary.

Genomic DNA from Bacillus cells is prepared as taught in CurrentProtocols In Molecular Biology. Vol. 1, edited by Ausubel et al., JohnWiley & Sons, Inc., 1987, Chapter 2.4.1. Generally, Bacillus cells froma saturated liquid culture are lysed and the proteins removed bydigestion with proteinase K. Cell wall debris, polysaccharides, andremaining proteins are removed by selective precipitation with CTAB, andhigh molecular weight genomic DNA is recovered from the resultingsupernatant by isopropanol precipitation. If exceptionally clean genomicDNA is desired, an additional step of purifying the Bacillus genomic DNAon a cesium chloride gradient is added.

After obtaining purified genomic DNA, the DNA is subjected to Sau3Adigestion. Sau3A recognizes the 4 base pair site GATC and generatesfragments compatible with several convenient phage lambda and cosmidvectors. The DNA is subjected to partial digestion to increase thechance of obtaining random fragments.

The partially digested Bacillus genomic DNA is subjected to sizefractionation on a 1% agarose gel prior to cloning into a vector.Altematively, size fractionation on a sucrose gradient can be used. Thegenomic DNA obtained from the size fractionation step is purified awayfrom the agarose and ligated into a cloning vector appropriate for usein a host cell and transformed into the host cell.

EXAMPLE II

Detection of Gram Positive Microorganisms

The following example describes the detection of gram positivemicroorganism MP.

DNA derived from a gram positive microorganism is prepared according tothe methods disclosed in Current Protocols in Molecular Biology, Chap. 2or 3. The nucleic acid is subjected to hybridization and/or PCRamplification with a probe or primer derived from MP.

The nucleic acid probe is labeled by combining 50 pmol of the nucleicacid and 250 mCi of [gamma ³²P] adenosine triphosphate (Amersham,Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN®, Boston Mass.).The labeled probe is purified with Sephadex G-25 super fine resin column(Pharmacia). A portion containing 10⁷ counts per minute of each is usedin a typical membrane based hybridization analysis of nucleic acidsample of either genomic or cDNA origin.

The DNA sample which has been subjected to restriction endonucleasedigestion is fractionated on a 0.7 percent agarose gel and transferredto nylon membranes (Nytran Plus, Schleicher & Schuell, Durham N.H.).Hybridization is carried out for 16 hours at 40 degrees C. To removenonspecific signals, blots are sequentially washed at room temperatureunder increasingly stringent conditions up to 0.1×saline sodium citrateand 0.5% sodium dodecyl sulfate. The blots are exposed to film forseveral hours, the film developed and hybridization patterns arecompared visually to detect polynucleotide homologues of B. subtilis MP.The homologues are subjected to confirmatory nucleic acid sequencing.Methods for nucleic acid sequencing are well known in the art.Conventional enzymatic methods employ DNA polymerase Klenow fragment,SEQUENASE®D (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest.

Various other examples and modifications of the foregoing descriptionand examples will be apparent to a person skilled in the art afterreading the disclosure without departing from the spirit and scope ofthe invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

1-20. (Cancelled)
 21. An expression vector comprising a nucleic acidencoding a gram positive metalloprotease comprising the nucleotidesequence shown in SEQ ID NO:1.
 22. An expression vector comprising anucleic acid having at least 85% homology with nucleic acid shown in SEQID NO:1.
 23. A host cell comprising an expression vector according toclaim
 21. 24-30. (Cancelled)
 31. A host cell comprising an expressionvector according to claim 22.